Cell Wall Polysaccharides of Marine Algae

Abstract

Marine algae are interesting as a plentiful source of many bioactive compounds, including polysaccharides, which represent various structurally different polymers of high diversity in monosaccharide composition, absolute and anomeric configuration, glycosidic linkages, molecular mass, and the presence and distribution of various functional groups. These polysaccharides are used by algae as cell wall structural components or food reserve. Algal polysaccharides are highly indicative for main algal taxa. Sulfated galactans of periodical unit/linkage sequence (agars, carrageenans) are typical for red algae, alginates, and fucoidans for brown algae; sulfated glucuronoxylorhamnans (ulvans) and other sulphated glycans for green algae. Algal cell wall polysaccharides are extracted from the raw material and further purified by preparative chromatography and/or chemical treatment. Many algal polysaccharides are assigned as phycocolloids due to their good solubility in water and their ability to create colloid systems, including gels and films. They also demonstrate various biological activities (immunomodulation, antitumor, anticoagulant, antiviral, and many other activities), which are prerequisites of pharmaceutical and medicinal applications. Finally, initially inactive or weakly active natural algal polysaccharides can be partially degraded or structurally modified to be fitted to various medicinal applications.

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1 Overview

Marine algae are a plentiful source of many bioactive compounds. Among these chemical components, polysaccharides are of interest because of their physical and chemical properties, which are useful for food and medicinal applications. Algae synthesize various types of polysaccharides as cell wall structural components or energy reserves. Algal polysaccharides demonstrate a high specificity to raw algae, which has been used by phycologists to clarify the phylogenetic relationship between main algal taxa and higher (vascular) plants [1, 2, 3]. Several species of common marine algae that are the source for the isolation of polysaccharides for food and medicinal purposes are shown in Fig. 22.1.

Fig. 22.1a–i

Marine algae producing polysaccharides of medicinal importance: (a)  Enteromorpha (Ulva) linza (Ulvales, Chlorophyta); (b) Capsosiphon fulvescens (Ulotrichales, Chlorophyta); (c)  Sargassum muticum (Fucales, Phaeophycea); (d) Ecklonia cava (Laminariales, Phaeophycea); (e)  Undaria pinnatifida (Laminariales, Phaeophycea); (f)  Laminaria japonica (Laminariales, Phaeophycea); (g) Carpopeltis affinis (Cryptonemiales, Rhodophyta); (h)  Chondrus ocellatus (Gigartinales, Rhodophyta); (i)  Scinaia japonica (Nemaliales, Rhodophyta). Algal photos were kindly provided by Dr. Myounglae Cho, East Sea Institute of the Korea Institute of Ocean Science and Technology (KIOSTI), Korea

1.1 Structure of Algal Cell Walls

Algal cell walls are composed of a highly integrated network of biopolymers, mainly polysaccharides, which interact with water, metal cations, and other molecules [2]. Structural specificities, including substitution patterns and degrees, are prerequisites of the physical and functional properties of cell wall polysaccharides in marine algae. Crystalline and fibrous parts (cellulose, xylans, hemicelluloses, etc.) are embedded in a gel-like matrix made of carboxylic and/or sulfated polysaccharides. Other biopolymers (proteins, proteoglycans, and polymeric phenolics) may also participate in cell wall formation.

1.2 Polysaccharides as Taxonomic Markers of Marine Algae

The great variability of cell wall polysaccharides in marine algae is determined by the specie/taxa [3, 4, 5, 6, 7], anatomical part of the alga [7], the developmental and life-cycle stage [10, 11, 7, 8, 9], and season and habitat [12, 13]. Algae represent a diverse group of photosynthetic eukaryotes with a wide range of cell wall types [2]. Besides pigmentation and food reserve specificity, matrix cell wall polysaccharides are useful as structural and taxonomical markers of marine algae. Cell wall polysaccharides from various marine algae of three main divisions, i. e., Clorophyta (green algae ), Rhodophyta (red algae ), and Phaephyta (brown algae ), are summarized in Table 22.1.

Tab. 22.1 Major cell-wall polysaccharides in the main groups of algae [1, 2, 3]

Green algae produce various sulfated and/or carboxylic cell wall polysaccharides of specific structure. Red algae have complex composite cell walls made of cellulose, xylan, or mannan fibrils and sulfated galactans (agars, carrageenans) as the main matrix components [14]. Brown algae produce cell walls containing cellulose fibrils and two main types of matrix polysaccharides (alginates, fucoidans). Cell walls of all marine algae are rich with sulfated matrix polysaccharides. This is an adaptation to marine habitats that has also been observed for many other marine organisms, including angiosperms and invertebrates [15]. There are two groups of algal sulfated polysaccharides (GlossaryTerm

SPS

) [16], i. e., (i) uronic acid-rich (GlossaryTerm

UA

: uronic acid) and (ii) GlossaryTerm

UA

-limited ones. In the green algae of Ulvophyceae [3], the first group is represented by ulvans (genera Ulva, Monostroma, Gayralia, and Acetabularia) [5]; the second one includes sulfated ($1\to 3$)-β-d-galactans [17, 18, 19, 20, 21, 22], ($1$ $\to$ $3$)-β-l-arabinopyranans [23], and β-($1\to 4$)-d-mannans [24] (genera Caulerpa, Codium, and Bryopsis). In addition, alkali-soluble linear ($1\to 4$)-β-d-glucuronan found in some green algae plays the role of hemicellulose [25]. Red algae contain linear and branched sulfated galactans having no uronic acids. By contrast, brown algae have specific kinds of carboxylic and sulfated polysaccharides, namely alginates and fucoidans; some of the latter ones may also contain galactose or glucuronic acid residues. More complex highly branched sulfated heteropolysaccharides (xylofucoglucans, xylofucoglucuronans, etc.) of brown algae were assigned as hemicelluloses.

2 Structural Diversity of Algal Polysaccharides

Algal cell wall polysaccharides are structurally variable polymers showing significant differences in molecular mass, monosaccharide composition, absolute and anomeric configuration of sugar units, glycosidic linkages, presence and distribution of functional groups (carboxylates, methyl ethers, acetyl esters, sulfate semi-esters, 3,6-anhydro and others). There are linear and branched polysaccharides, the latter may have stubs, longer side chains or even very complex branching with defined core, inner and peripheral parts. Basic structure of common algal polysaccharides is demonstrated in Fig. 22.2. Structural variability of specific algal polysaccharides is reviewed below.

Fig. 22.2a–h

Basic structures of marine algal polysaccharides: (a)  alginate (G – guluronic acid, M – mannuronic acid); (b)  κ-carrageenan; (c)  agarose-6-sulfate; (d,e) fucoidans; (f–h) ulvans

2.1 Alginates

Alginates are matrix cell wall polysaccharides of brown algae (Phaeophyceae). They are linear co-polymers of ($1\to 4$)-linked units of β-d-ManpA and its C-5 epimer α-l-GulpA. These uronic acids are distributed in three types of blocks containing (i) only β-d-ManpA (GlossaryTerm

M

-blocks, GlossaryTerm

M

: mannuronic acid), (ii) α-l-GulpA (GlossaryTerm

G

-blocks, GlossaryTerm

G

: guluronic acid), or (iii) alternating sequences of both these residues (GlossaryTerm

MG

-blocks) [44]. In algal cells alginates are synthesized as homomannuronan polymeric chains and then β-d-ManpA units are partially converted to α-l-GulpA ones by enzyme C-5 epimerase. This conversion is irreversible and both types of blocks are formed. The ${}^4{\mathrm{C}}_1$ and ${}^1{\mathrm{C}}_4$ conformations were confirmed for β-d-ManpA and α-l-GulpA alginate units, respectively [30, 45]. In the homopolymeric blocks the units are bound with diequatorial (M-blocks) and diaxial (G-blocks) glycosidic linkages, while in the alternating sequence the units are bound by mixed equatorial-axial (GlossaryTerm

MG

) and axial-equatorial (GlossaryTerm

GM

/GlossaryTerm

MG

 – alternating blocks in alginates) linkages (Fig. 22.2a). The homopolymeric blocks form helical structures stabilized by intra-molecular hydrogen bonds involving hydroxyls, ring oxygens, and/or carboxyls; the GlossaryTerm

MG

-blocks take up disorder conformations. These helices demonstrate different stiffnesses: G-blocks look like buckled ribbons and they are stiffer than flat-ribbon M-blocks [41, 46, 47]. The GlossaryTerm

M/G

ratio (molar ratio between mannuronic and glucuronic acids in alginates) and distribution of co-monomer residues along an alginate chain depend on the source and geographic location of raw algal material, season of collection, the age of the parts of the plant, and extraction methodology used. The M/G ratio of alginates originating from Indian algae (the coast of Kilakarai, the Gulf of Mannar, South India) was reported to be maximal in summer and minimal in premonsoon seasons; this value also depends on the anatomical part of algal thalli used for isolation [12, 13]. The high variability of the M/G ratio for alginates from various brown algae is illustrated in Table 22.2. The block composition of alginates was investigated by a partial acidic hydrolysis ($0.3{\mathrm{molL}}^{-1}$ HCl, $100\text{°C}$, $2\mathrm{h}$) followed by precipitation of the resistant fragments [11, 32]. Strong acidic hydrolysis of alginates from Laminaria brasiliensis gave rise mainly to M and G-oligosaccharide blocks, indicating preferential hydrolysis of glycosidic bonds in the GlossaryTerm

MG

-regions [42]. Two types of alginates were defined based on the distribution of monomers [32], i. e., those containing (i) many G-blocks, less GlossaryTerm

MG

-blocks, and even less M-blocks (GlossaryTerm

GG

$>$ GlossaryTerm

MM

$>$ GlossaryTerm

MM

), and (ii) a small proportion of GlossaryTerm

GG

and near equal proportions of GlossaryTerm

MG

and M-blocks (GlossaryTerm

GG

$<$ GlossaryTerm

MG

$\approx$ GlossaryTerm

MM

). Alternatively, algal alginates could be classified as being high-M, low-M (or high-G), or intermediate type [27]. Well-defined GlossaryTerm

MG

-blocks were detected only for the intermediate alginates, whereas the high-M and low-M alginates consist mainly of homopolymeric blocks (GlossaryTerm

GG

, GlossaryTerm

MM

), which are almost directly connected and contain few or no heteropolymeric blocks. It was also found that young algal tissues are rich in M-blocks, and that the difference between the alginates from different species was mainly due to the contribution of the older parts of thalli [11].

Tab. 22.2 M/G values obtained for alginates from various algal sources

2.2 Sulfated Galactans

Sulfated galactans are common cell wall matrix polysaccharides of green (Clorophyta) and red (Rhodophyta) algae. They are polymers of partially sulfated β-d- sand/or α-d(l)-galactopyranosyl (Galp) units.

Sulfated galactans from cell walls of green algae (genera Codium and Bryopsis) were reported to be both homo and heteropolysaccharides, depending on the species [17, 19, 20, 22, 51]. Polysaccharides isolated from Codium isthmocladum [20] and Codium yezoense [17, 52] are sulfated homogalactans containing similar backbones, composed mainly of ($1\to 3$)-linked β-d-Galp units and short oligosaccharide side chains bound at O-6. The units were sulfated mainly at O-4 and rarely at O-6, and nonreducing terminal residues were pyruvylated forming cyclic 3,4-ketals. Sulfated galactan isolated from Bryopsis plumosa (Bryopsidales, Chlorophyta) consist of a ($1\to 3$)-linked β-d-Galp partially sulfated on O-6 and substituted with pyruvic acid, forming 4,6-ketals [19]. A branched sulfated ($1\to 3$), ($1\to 6$)-β-d-galactan was isolated from Codium fragile. This polysaccharide has a ($1\to 3$)-linked partially 4-O-sulfated β-d-Galp backbone with some 3,4-pyruvated β-d-Galp stubs at O-6 positions [21]. By contrast, galactans isolated from Codium fragile [51] and Codium cylindricum [22] contain other sugar units – arabinose and glucose, respectively. Nevertheless, the $\to$3)-β-d-Galp4S-(1$\to$ units still predominate in these polysaccharides. Water-soluble highly sulfated arabinans, galactans, and/or arabinogalactans were isolated from Codium fragile and Codium vermilara [18]. These polysaccharides are composed of ($1\to 3$)-linked β-d-Galp and β-l-Arap residues; they are partially substituted with pyruvic acid ketals.

Red algal cell walls contain ($1\to 3$), ($1\to 4$) – mixed linkage α, β-d(l)-galactans. Most of these polysaccharides are nonbranched. Their backbone is made of disaccharide repeating fragments of alternating ($1\to 3$)-β- and ($1\to 4$)-α-linked Galp. The former units (G) always have d-configuration, whereas the latter ones (d or l) are present as the d- (the carrageenan family) or l- (the agaran family) enantiomers (Fig. 22.2b,c). Sulfate hemiesters, methyl ethers, and pyruvic acid are commonly bound to these polysaccharides. Sulfate and/or occasionally methyl groups may occur at the O-2 and/or O-4 position(s) of the ($1\to 3$)-β-linked Galp units and at O-2, O-3-, and/or O-6 position(s) of the ($1\to 4$)-α-linked Galp units; pyruvic acid may be attached to ($1\to 3$)-β-d-Galp residues as ketals at O-4 and O-6 positions [81, 82]. The 3,6-anhydro derivatives of d(l) units may be present in the backbone of carrageenose (agarose) after elimination of sulfate from O-6 by enzymatic or alkali treatment. Agarose, a simplified structure of agar, is a sequence of identical disaccharide (agarobiose) fragments without sulfates. By contrast, real agars have some α-l-Galp6S (porphyran), pyruvic acid 4,6-ketals, O-methyl, or sulfate groups attached to β-d-Galp residues [81, 83]. Similarly, carrageenans may have a structure described by one of the defined disaccharides (carrabioses) and thus are classified by Greek prefixes according to their structural specificity, i. e., sulfation patterns and the presence of 3,6-anhydro groups (Table 22.3). More often, however, they contain several types of disaccharide fragments, i. e., hybrid types (Table 22.4). Structural and conformational analysis of $\kappa /\iota$ hybrid carrageenans from Sarcothalia crispala, Mazaella laminarioitles, and Chonrlrus crispus showed that they are not a mix of pure carrageenans but have a mixed chain containing both κ and $\iota$-disaccharide fragments [84]. Gametophyte and sporophyte generations of the algal biological cycle contain carrageenans of different structure, especially sulfate and 3,6-anhydride groups. Carrageenans of Phyllophoraceae and Gigartinaceae were described to be hybrids of κ, $\iota$, and ν forms for gametophytes and rather λ subfamily forms (λ, ξ, and π) for tetrasporophytes [10, 64, 68, 69, 85, 86, 9]. Carrageenans usually contain more sulfates than agarans, while the latter often contain more methyl and glycosyl groups (Table 22.3). The structural variability of red algal galactans, especially of their substitution patterns, has been described for different species, harvesting location, or seasons. Sometimes the structure of red algal galactans ($\alpha /\beta$ anomers, d/l enantiomers, sequence of linkages, substitution patterns, etc.) deviates from the defined structural types of agars and carrageenans. Other sugars (Xyl, Glc, GlcA, Ara, or Man) are bound to some of them as backbone or side chain parts [87]. There are also some polysaccharides, named dl-galactan hybrids, having both d and l-units distributed within the same polysaccharide chain [88]. The real structure of red algae sulfated galactans is probably a result of the following stepwise biosynthesis: (i) formation of the regular polymer backbone, (ii) sulfation, methylation, and/or pyruvation of specific hydroxyls, and (iii) enzymatic conversion of some ($1\to 4$)-α-linked Galp6S residues to 3,6-anhydro derivatives. According to this sequence, porphyran is a precursor of agarose, and, similarly, it is true for the pairs of carrageenans. Formation of 3,6-anhydrogalactose, similar to the final step of biosynthesis, is possible by the treatment with heated dilute alkali on the corresponding precursor. The corallinans, which are agar-like branched sulfated and methylated galactans from red algae of the order Corallinales, have (a) an agaran backbone, (b) a β-d-Galp unit almost completely substituted at O-6 mostly with β-d-xylosyl side chains and less by sulfate, and (c) α-l-Galp units partly substituted by methoxyl and sulfate groups at O-2 [89]. In addition, the latter units can be substituted at O-3 or O-6 with partially O-methylated α-d- or α-l-Galp side chains (Table 22.5). Polysaccharides from various algae may differ in the position of these branches. There is a balance between the hydrophobic (methoxyl) and the hydrophilic (sulfate) groups, so the algal enzymatic system equilibrates their proportions to fulfill their biological design [90]. Decalcified tissue from Calliarthron cheilosporioides contains sulfated galactan, highly methoxylated but lowly glycosylated with xylose at O-6 of the β-d-Galp units [91]. By contrast, calcified segments of this alga produce xylogalactans with high levels of xylose and low levels of 6-O-methyl ethers. Therefore, decalcification of algal tissues led to replacing xylosyl branches by methyl groups. These structural changes are caused by specific methoxyl transferase blocking xylosylation in decalcified tissue.

Tab. 22.3 Disaccharide repeating structure and abbreviation [14] of some agarans and carrageenans

Tab. 22.4 Hybrid carrageenans and dl-galactans

Tab. 22.5 Side chain positions in corallinans and other agar-like branched sulfated xylogalactans

2.3 Fucoidans

Fucoidans are sulfated matrix cell wall polysaccharides of brown algae (Phaeophyceae). Simple fucoidans are sulfated homofucans, i. e., polysaccharides consisting of sulfated fucose only. However, in some reports this term is used to describe complex fucose-containing heteropolysaccharides. The chemical composition and structure of fucoidans are complex and variable, reflecting the differences in biosynthesis [152, 6]. Very often the exact structure of these polysaccharides remains unclear even when modern methods of structural analysis like selective enzymatic cleavage or correlation GlossaryTerm

NMR

analyses are applied. This problem is caused by significant irregularities in the structure of algal fucoidans, including the presence of numerous minor sugar and nonsugar components, random sulfation, and/or acetylation [153, 6]. There are two main types of backbone structure found in fucoidans [152, 153, 154] (Fig. 22.2d,e, Table 22.6). The first type of fucoidan backbone structure consisting of ($1\to 3$)-linked partially or completely sulfated α-l-Fucp units revealed fucoidans from orders Ectocarpales and Laminares: Adenocystis utricularis [141], Analipus japonicus [114], Chordaria flagelliformis [140], Chorda filum [127], Ecklonia kurome [123], and Saccharina latissima [128]. The second type is characterized by alternating the ($1\to 3$) and the ($1\to 4$)-linked α-l-Fucp sequence. This backbone structure is common for fucoidans from Stoechospermum marginatum (order Dictyotales) [116] and from algae of the order Fucales, i. e., Ascophyllum nodosum [117], genera Fucus [118, 119, 120] and Sargassum [121]. Single α-l-Fuc stubs or short fuco-oligosaccharide sequences are present in many fucoidans as side chains attached mainly at O-2 of the backbone units. Structurally different fucoidans are commonly produced by the same algal species. For example, fucoidan isolated from Ascophyllum nodosum has a linear structure of alternating ($1\to 3$) and ($1\to 4$)-linked sulfated α-l-Fucp [116]. By contrast, another fucoidan isolated from this alga is defined as a highly-branched polysaccharide with mainly ($1\to 3$) and a few α-($1\to 4$)-linked sulfated α-l-Fuc in the core region; branch points are at O-2 position of the ($1\to 3$)-linked backbone units [115]. Three fucoidans from Sargassum hornery have a different backbone structure: ($1\to 3$), ($1\to 4$), and mixed ($1\to 3$),($1\to 4$)-linked α-l-fucans; the latter polysaccharide was nonsulfated [122].

Tab. 22.6 Basic structures of sulfated fucans (fucoidans) from brown algae

Some fucoidans were identified as not true fucans but rather complex heteropolysaccharides containing fucose as the major or minor component of the backbone (core), stubs, or longer side chains [152, 153, 154]. The structural features of these polysaccharides are summarized in Table 22.7. The core of such fucoidans is commonly built of alternating hexuronic acids–hexose sequences. This backbone is very stable and other sugars including sulfated or nonsulfated α-l-Fucp can be attached to it as stubs or longer side chains. The complete highly branched molecule is thus structurally similar to rhamnogalacturonans of higher plants and may perform similar functions in algal cell walls. For example, highly branched fucoidan ($92.7\mathrm{kDa}$) isolated from Hizikia fusiforme contains a fucose-free core composed of the alternating sequence $\to$ $2$)-α-d-Manp6S-($1\to 4$)-β-d-GlcpA-($1$ $\to$ with a little amount of $\to$ $4$)-β-d-Galp-($1$ $\to$ [146]. The α-d-Manp6S units are sulfated or glycosylated (branching points) at the O-6 position. Side chains are formed by α-d-Xylp, sulfated α-l-Fucp, fuco and xylo-oligosaccharides, and longer $\to$ $6$)-β-d-Galp3S-($1$ $\to$ chains. Another variant of the core was found in fucoidan of Padina gymnospora [144]. The backbone of this polysaccharide is glucuronan, consisting mainly of ($1\to 3$)- or ($1\to 4$)-linked β-d-GlcA. Ascophyllan, a branched xylofucoglycuronan isolated from Ascophyllum nodosum. It has a backbone made of uronic acid, mainly ($1\to 4$)-linked β-d-ManpA, and branches containing sulfated α-l-Fucp and β-d-Xylp [145]. Sargassan, a complex polysaccharide from Sargassum linifolium, has a glucuronomannan core with internal side chains of ($1\to 4$)-linked sulfated β-d-GlcA and peripheral side chains made of sulfated α-l-Fucp and β-d-Xylp [148, 149, 150, 151].

Tab. 22.7 Basic structures of fucose containing heteroglycans from brown algae

Complex highly branched polysaccharides have been isolated from Sargassum stenophyllum [147]. One of these had a linear galactomannan core formed by ($1\to 6$)-β-d-Galp and/or ($1$ $\to$ $2$)-β-d-Manp units with side chains containing β-d-Xylp, ($1\to 3$)- and/or ($1\to 4$)-linked α-l-Fucp, ($1\to 4$)-linked α-l-GlcpA, and α-d-Glcp. Two polysaccharides obtained from Adenocytis utricularis were identified as galactofucan and glycuronofucan [141].

2.4 Ulvans

Ulvans are principal matrix cell wall polysaccharides of green algae, genus Ulva [155]. The composition of these polysaccharides varied according to species, harvest season, growth conditions, and method of isolation [156, 5]. Four monosaccharides, i. e. α-l-Rhap, β-d-Xylp, β-d-GlcpA and α-l-IdopA, and sulfate are the main constituents of ulvans [155, 156]. Like in the case of alginates, these two uronic acids are C-5 epimers. All these types of sugar units are commonly arranged in the backbone, while single β-d-GlcpA can also constitute the side chains. The basic structure of ulvans is described by several repeating disaccharide fragments called ulvanobiuronic acids or ulvanobioses. Ulvanobiuronic acids (Fig. 22.2f,g) contain α-l-Rhap and uronic acid residues, while ulvanobioses contain β-d-Xylp instead of uronic acid (Fig. 22.2h). Ulvans isolated from several Ulvales species are composed of variable proportions of these disaccharides. Table 22.8 summarizes various oligosaccharides defined as structural fragments of ulvans.

Tab. 22.8 Basic structures of ulvans from Ulva sp. (after [5])

2.5 Other Polysaccharides

Cell walls of many green algae contain alkali soluble linear ($1\to 4$)-β-d-glucuronans [157, 158, 159, 25]. Sulfated rhamnans have been isolated from Monostroma nitidum and Monostroma latissinum (Ulotrichales, Chlorophyta) [160, 161, 162, 163, 164, 165, 25] and from Gayralia oxysperma (Ulvales, Chlorophyta) [166, 25]. Similar polysaccharides but in smaller amounts have been detected in Bryopsis plumosa (Bryopsidales, Chlorophyta) [19]. These polysaccharides have mainly ($1\to 2$)-linked, but also ($1\to 3$) and/or ($1\to 4$)-linked partially sulfated α-l-Rhap units. Moreover, they have single stubs of uronic acid residues as side chains. Rhamnan sulfate from Monostroma nitidum consists of ($1\to 3$)-linked α-l-Rhap residues, some of which are sulfated mainly at O-2 [162]. Minor amounts of internal ($1\to 2$)-linked and branched α-l-Rhap linkages have also been found. Branched sulfated glucorhamnan has been isolated from the same alga [162]. This polysaccharide is mainly composed of ($1\to 2$) and ($1\to 3$)-linked α-l-Rhap ($1:2$) partially sulfated at the O-3 and O-2 positions, respectively. In addition, ($1\to 4$)-linked β-d-Glcp fragments are bound at O-2 of some ($1\to 3$)-linked backbone units. By contrast, polysaccharide from Monostroma latissinum is defined as highly sulfated rhamnan composed mainly of ($1\to 2$)-linked α-l-Rhap residues with sulfate groups substituted at O-3 and/or O-4 [163]. Sulfated heterorhamnan isolated from Gayralia oxysperma has ($1\to 3$) and ($1$ $\to$ $2$)-linked α-l-Rhap units in the backbone [166]. The latter units are partially substituted at C-3 by side chains containing single GlcpA, GalpA or Xylp. Both types of Rhap units are partially sulfated at O-2 and/or O-4; side chain uronic acids are also sulfated but only at O-2.

Sulfated xylomannans, linear or containing single stubs of β-d-Xylp, or 3-O-methyl-β-d-Xylp at O-2, have been described for many red algae. Branched sulfated xylomannans ($33$–$222\mathrm{kDa}$) have been isolated from two species of the genus Chondrophycus (Ceramiales, Rhodophyta) [167]. These polysaccharides consist of ($1\to 4$)-linked β-d-Manp2S backbone carrying stubs ($15$–$25\%$) of single β-d-Xylp ($70$–$80\%$) and β-d-Manp2S ($20$–$30\%$). Several fractions from Nothogenia fastigiata (Nemaliales, Rhodophyta) have been identified as xylomannans with a backbone of ($1\to 3$)-linked α-d-Manp sulfated at the O-2 and O-6 positions and single stubs of β-d-Xylp at O-2 [168, 169]. Sulfated xylomannan ($150\mathrm{kDa}$) has been isolated from Sebdenia polydactyla (Sebdeniales, Rhodophyta) [170]. This polysaccharide contains a backbone of ($1\to 3$)-linked α-d-Manp units, partially substituted with a single stub of β-d-Xylp at O-6. The main polysaccharide fraction isolated from Nemalion helminthoides (Nemaliales, Rhodophyta) has been identified as ($1\to 3$)-α-d-mannan sulfated at the O-4 and O-6 positions; other fractions are similar mannans but they contain single stubs of β-d-Xylp [171, 172]. Sulfated arabinopyranan has been isolated from the green alga Enteromorpha clathrata [173]. The backbone of this polysaccharide is mainly composed of ($1\to 4$)-linked β-l-Arap residues, partially sulfated at the O-3 position. Two sulfated polysaccharides, i. e., arabinan and arabinogalactan, have been isolated from Codium dwarkense (Bryopsidales, Chlorophyta) [174]. Two water-soluble sulfated polysaccharides from Codium vermilara (Bryopsidales, Chlorophyta) have been identified as (i) partially 2-O-sulfated ($1\to 4$)-β-d-mannan and (ii) ($1\to 3$)-β-l-arabinopyranan highly sulfated at the O-2 and O-4 positions [23, 24].

3 Isolation from Algal Raw Material

Crystalline, fibrilar, and matrix polysaccharides of algal cell walls can be successfully isolated from raw material by subsequent extractions. The isolation is based on the difference in solubility of these polysaccharides in various media at neutral, acidic, or alkali conditions. Usually matrix polysaccharides are well soluble in hot water, hemicellulose components are soluble in alkali solutions, while crystalline cellulose and, in some cases, xylans or mannans, remain insoluble at these conditions. Before subsequent extraction of polysaccharides, raw material is usually washed with organic solvents to remove lipids, pigments, and other small molecules, and then washed with distilled water to remove small hydrophilic molecules. Finally, raw material is dried and milled.

3.1 Extraction Procedures

The subsequent extraction steps vary depending on whether the polysaccharides contain sulfate and/or carboxylic groups. Routine procedures of common extraction of polysaccharides from various algae are outlined in Table 22.9. Extraction with cold water is followed by extractions with hot water and alkali solutions at different pH and temperatures [175]. This approach could be defined as universal because it is effective for the isolation of major polysaccharides from red algae (sulfated galactans, agars, and carrageenans), brown algae (sulfated fucoidans and xylans), and green algae (heteroglycuronans, ulvans). Neutral sugar (GlossaryTerm

NS

) and GlossaryTerm

UA

compositions vary significantly for crude water and alkali extracts, so it is possible to separate water and alkali soluble polysaccharides. For example, sulfated galactans predominate in water extracts, while floridean starch predominates in the alkali extracts. Heterofucan was assigned as the major polysaccharide in extracts from brown algae; however, alginates were not analyzed in this report. Alternatively, a combination of other media can be used for extractions.

Tab. 22.9 Extraction of marine algal polysaccharides

In other work [176], brown algae (Ascophyllum nodosum, Fucus vesiculosus, and Saccharina longicruris) were subsequently treated with selective extraction media at $70\text{°C}$ to obtain three fractions: fucoidan in a mixture with laminaran ($2\%$ aq. ${\mathrm{CaCl}}_2$), fucoidan ($0.01{\mathrm{molL}}^{-1}$ aq. HCl) and alginate ($3\%$ aq. ${\mathrm{Na}}_2{\mathrm{CO}}_3$). Three different ways of sodium alginate isolation from brown algae Macrocystis pyrifera (Table 22.10) were compared in [49]. Acid pre-treatment was used in order to eliminate polyvalent cations prior to the extraction.Alginate was then solubilized under basic conditions and purified in different ways. Three methods of precipitation/purification (ethanol, HCl, and ${\mathrm{CaCl}}_2$ routes) were used to study the influence of process conditions on the final products. Both HCl and ${\mathrm{CaCl}}_2$ routes give low $M_{\mathrm{w}}$ alginates of poor mechanical properties owing to the partial acidic hydrolysis of the polymeric chain by the treatment with $1{\mathrm{molL}}^{-1}$ HCl. By contrast, the ethanol route showed the highest yield and good rheological properties of the products. The diluted acid pre-treatment improved the yield of the ethanol route, avoiding the acidic hydrolysis of alginate.

Extraction of agars and carrageenans needs different conditions because these polysaccharides have a marked difference in sulfate contents [178]. Carrageenophytes (Mastocarpus stellatus, Chondrus crispus, Calliblepharis jubata, Chondracanthus acicularis, and Chondracanthus teedei) have been used for carrageenan extraction by alkali treatment ($1{\mathrm{molL}}^{-1}$ of NaOH, $80\text{°C}$, $3\mathrm{h}$), whereas agars have been isolated from agarophyte (Gracilaria gracilis) by hot water extraction ($90\text{°C}$, $4\mathrm{h}$). Two major cell-wall polysaccharides have been extracted from the green seaweed Ulva rigida sequentially with oxalate and KOH containing media [157]. Sulfated glucuronorhamnoxylan (ulvan) has mainly been extracted with oxalate, while two hemicelluloses, i. e., ($1\to 4$)-β-d-glucuronan and glucoxylan, have been isolated at alkali conditions.

Kinetic modeling (GlossaryTerm

KM

) is often used for optimization of extraction procedures and to obtain good quality products. For example, a kinetic model has been proposed for the alkaline extraction of alginate from the fresh brown alga Laminaria digitata in order to predict the yield as a function of the stirring level and the size of algae pieces [179]. Such models may help to reduce the extraction time of high-quality alginate production. The proposed model was defined as a first step in the more complete extraction kinetic model, taking into account the effect of temperature. The effects of various variables (alkali treatment, relative amount of water, soaking/extraction time, and temperature) have been investigated on agar extraction from the red alga Gracilaria cliftonii [180]. The agar yield was significantly affected by all these variables.

Tab. 22.10 Three ways of sodium alginate isolation from algal material according to [49]

Response surface methodology (GlossaryTerm

RSM

) has been used to determine the optimum extraction conditions for yield and further parameters of agar extraction from Hydropuntia cornea [177] and carrageenan extraction from Kappaphycus alvarezii [181]. This method explores the relationships between several explanatory (reaction conditions of designed experiments – temperature, extraction time, pH, concentration of reagents, etc.) and response (yield, molecular weight, structural features, and physical properties of extracted polysaccharides) variables [182]. Among the parameters of reaction conditions, the extraction temperature is supposed to be the most important for increasing of the yield. Extraction procedures can be also optimized by the assistance of other additional factors like extrusion, microwaves (GlossaryTerm

MW

), or specific enzymes. Alkaline extraction of alginates from Laminaria digitata has been modified by reactive extrusion, which enables process time savings, yield increase, and improvement of the rheological properties of the products [183].

Microwave-assisted extractions (GlossaryTerm

MAE

) of algal polysaccharides have been tested and optimized [183]. A combination of mathematical and statistical techniques allowed us to determine the optimal experimental conditions for GlossaryTerm

MAE

extraction of agar from Gracilaria vermiculophylla. Enzyme-assisted extraction has also been used in the processing of marine algae [184] using endoprotease (Neutrase) for enzymatic digestion. A novel enzymatic extraction of marine algal polysaccharides with cellulase using GlossaryTerm

RSM

to search for the optimal conditions (enzyme concentration, ratio of medium/raw material, and extraction time) has been developed to reach high yields [185].

3.2 Purification of Crude Extracts

The crude extracts of algal polysaccharides are usually concentrated, dialyzed against distilled water, and then freeze-dried or precipitated with an excess of ethanol. Crude extracts usually contain a mixture of two or more polysaccharides, for example, pairs of carboxylic and sulfated ones like an alginate/fucoidan mixture from brown algae or an ulvan/glucuronan mixture from green algae. These components are effectively separated by changing the pH or by treatment with ${\mathrm{Ca}}^{2+}$ cations [49]. Alginic acids are insoluble due to protonation of carboxyls. In addition, G-blocks in alginates are able to form egg-box complexes with ${\mathrm{Ca}}^{2+}$, which leads to calcium alginate precipitation. By contrast, fucoidans remain soluble at low pH and weakly interact with divalent cations. Glucuronans are often co-extracted together with ulvans from the cell wall of Ulva sp. with the use of hot water that often contains a calcium chelating agent such as sodium oxalate; ion-exchange chromatography has been used to eliminate glucuronans from the ulvan preparations [157, 158].

By contrast to ulvans, ($1\to 4$)-β-d-glucuronans are insoluble at lower pH. Based on this difference, acidic precipitation (pH $2$) has been used to separate ulvan and glucuronan from the hot oxalate buffer extract ($0.05{\mathrm{molL}}^{-1}$, pH $6$, $90\text{°C}$, $3\mathrm{h}$) of Ulva lactuca [159]. The high amount of uronic acids ($94\%$ m/m) and the absence of sulfate groups have confirmed the purity of precipitated glucuronan. Extracts of crude algal polysaccharide have further been fractionalized and/or purified on various preparative columns using gel permeation and/or ion exchange chromatography with isocratic or gradient elution [173, 83]. These procedures lead to separation of polysaccharides according to their specific retention times and to removal of impurities like proteins, phenolics, or other polysaccharide admixtures. Proteins are also removed by the treatment with various chemical reagents or proteases; phenolics by the treatment with oxidative agents. Fucoidan was purified from its natural complex with polyphenols by the treatment with $5\%$ ${\mathrm{H}}_2{\mathrm{O}}_2$ (pH $8.3$–$8.7$, $18\text{°C}$), and this treatment did not lead to marked desulfation of the polysaccharide [186, 83]. Fucoidans treated with ${\mathrm{H}}_2{\mathrm{O}}_2$ have not demonstrated any significant changes in backbones or sulfation degrees compared with the raw polysaccharide [187, 83]. However, a decrease in $M_{\mathrm{w}}$ is possible at higher reaction temperatures, ${\mathrm{H}}_2{\mathrm{O}}_2$ concentration, and reaction time.

4 Algal Polysaccharides such as Phycocolloids

Algal matrix cell wall polysaccharides can be assigned as phycocolloids (or phycohydrocolloids) because they are soluble in water and can create colloid systems in aqueous media or participate in their formation. Some of these polysaccharides can form gels and films at appropriate conditions; the films are commonly obtained by solution casting and solvent evaporation. The physical and chemical properties of phycocolloids, including gel formation, have been reviewed in connection with their structure [188]. The geometry of glycosidic linkages and the ability to form intra and intermolecular junctions are prerequisites of gel and film formation. These associations may be affected by (i) negatively charged groups (sulfates and carboxylates), which tend to expend a polysaccharide chain due to mutual electrostatic repulsion; (ii) various substituents (methyls, acetyls, pyruvate ketals, glycosylation, etc.), which cause steric hindrance and/or participate in various intra and intermolecular interactions; and (iii) dissolved small molecules or metal cations that influence the hydratation property of water. Conditions such as temperature, pH, and ionic strength may also influence gel formation.

4.1 Alginic Acid/Alginate Gels and Films

Alginates of alkali metals and alginic acids form transparent, flexible, and water soluble films by casting of their aqueous solutions. Films prepared from GulpA rich alginate demonstrate lower plasticity and have proved to be better moisture barriers than those prepared from ManpA rich alginate under the same conditions [189, 190]. Alginate films are effective cation exchangers. Their solubility may significantly decrease in the presence of multivalent metal cations [191, 192]. The ${\mathrm{Ca}}^{2+}$ and ${\mathrm{Zn}}^{2+}$ cations make alginate film water insoluble, increase its tensile strength, and decrease elongation. These effects are less pronounced for ${\mathrm{Cu}}^{2+}$ and ${\mathrm{Al}}^{3+}$ cations and little pronounced for ${\mathrm{Fe}}^{3+}$ and ${\mathrm{Mg}}^{2+}$ cations. Metal cations of smaller ionic radius cause tighter cross-linking of alginate films [193]. Cross-linking by ${\mathrm{Ca}}^{2+}$ produces significantly thicker and stronger, but less elastic alginate films; water vapor permeability decreases significantly only for highly cross-linking films [194]. The incorporation of multivalent metal cations into the alginate films may partially replace the weak polar interactions between polysaccharide chains by stronger cation mediated electrostatic junctions [195].

Alginates are able to form two types of gels: (i) alginic acid gel at acidic conditions (pH$<$p$K_{\mathrm{a}}$ of uronic carboxyls) and (ii) cross-linking thermostable gels at neutral conditions in the presence of ${\mathrm{Ca}}^{2+}$. Acidic gels are stabilized by intermolecular hydrogen bonds and the junction zones of these gels are probably composed of random aggregates [196]. G-blocks are the most important fragments for both acidic and ${\mathrm{Ca}}^{2+}$ gel formation, whereas M-blocks support only acidic gel formation. Alternating or random sequences destabilize alginic acid gel formation due to their lack of repeating structure. In the case of neutral gels, cations of other alkaline earth metals, excluding ${\mathrm{Mg}}^{2+}$ or other divalent metal cations, can be used instead of ${\mathrm{Ca}}^{2+}$. G-blocks, but not GlossaryTerm

GM

or M-blocks, of alginates interact strongly and cooperatively with these cations, forming egg-box type structures [197, 47]. ${\mathrm{Ca}}^{2+}$ ions can replace carboxylic protons in the G-blocks, which are zipped into cavities [198, 199]. The ${\mathrm{Ca}}^{2+}$ binding capacity does not depend on the GlossaryTerm

M/G

ratio of alginates, but GulpA exhibit much stronger affinity to these cations than ManpA [200]. In this model, the cations (eggs) are situated in the cavities inside puckered boxes formed by four GulpA units of the two superimposed chains. The individual boxes potentially possess oxygen ligands from ${\mathrm{COO}}^{-}$, 2-OH, 3-OH, O-5 pyranoid rings, and O-4 of glycosidic bonds in the G-blocks; some of these ligands directly interact with ${\mathrm{Ca}}^{2+}$, others participate in intra and intermolecular hydrogen bonds stabilizing the egg-box complex [201]. Based on GlossaryTerm

FTIR

(Fourier transformed infrared) spectroscopic analysis of ionic alginate gels with various divalent metal cations, different coordination geometry was proposed for the metal-carboxylate complexes in G-blocks (pseudo-bridged unidentate) and M-blocks (bidentate bridging) [202]. The strength of alginate gels depends on the GlossaryTerm

M/G

ratio and distribution of GlossaryTerm

GG

, GlossaryTerm

MM

, and GlossaryTerm

GM

-blocks in the polysaccharide chain [203, 204]. Alginates with a low GlossaryTerm

M/G

ratio and a high content of G-blocks form dense and brittle gels, whereas alginates with a high GlossaryTerm

M/G

ratio and a high content of M-blocks give more elastic gels [12, 13, 205].

There are three distinct and successive steps in the binding of ${\mathrm{Ca}}^{2+}$ to alginate with increased concentration of ${\mathrm{Ca}}^{2+}$ ions [206]: (i) interaction of ${\mathrm{Ca}}^{2+}$ with a single GulpA unit forming mono-complexes, (ii) propagation and formation of egg-box dimers via pairing of these mono-complexes, and (iii) lateral association of these dimers, generating egg-box multimers. In freshly prepared gels, junction zones are assumed to consist of egg-box dimers only. At higher ${\mathrm{Ca}}^{2+}$ concentration upon drying, these dimers may associate laterally into ordered domains (egg-box multimers) by different ways, including the most probable electrostatic interactions between ${\mathrm{Ca}}^{2+}$ and carboxylates [198, 207]. Lateral association of alginate chains is reduced by removing ${\mathrm{Ca}}^{2+}$ excess from the gel beads in a washing step prior to air drying. The egg-box multimers are disrupted by noncross-linking cations (${\mathrm{Na}}^{+}$, ${\mathrm{K}}^{+}$ or ${\mathrm{Mg}}^{2+}$), which replace the ${\mathrm{Ca}}^{2+}$ bound to the alginate. The result is swelling of the alginate gel. ${\mathrm{Ca}}^{2+}$ alginate gelation has been described as random cross-linking of multifunctional monochains [208]. Alginate gels showed strain-hardening behavior at large deformations, which was explained by (i) the re-orientation of chain segments longer than the distance between cross-links [209], (ii) structure densification or highly cross-linked polymer systems [205], and (iii) the deformation of rod-like junction zones [210].

4.2 Gels and Films Based on Sulfated Galactans

Some red algal sulfated galactans (agars and carrageenans) are able to form films and thermally reversible hydrogels by cooling of their hot aqueous solutions. Agar films demonstrate a lower tensile strength, water vapor permeability, swelling ratio, and water solubility than κ-carrageenan films. Each property of the mixture agar/κ-carrageenan films varies depending on the ratio of each component [211]. Gelation of sulfated galactans is a complex process that depends on temperature, structure, and concentration of polysaccharide, and on the presence of metal cations, which can induce conformational changes in the polymer chain. As a result, the initial coil-to-helix transition is followed by subsequent aggregation of these helices [212, 213, 214, 215].

The final gel structure is the result of mutual interactions between conformational transition, molecular cross-linking, and phase-separation processes [216]. A honeycomb-like network has been observed for aqueous solutions both of agars and carrageenans at various temperatures. Its structure depends on the gelling stage, the structural type (d- or l- absolute configuration of the 3,6-anhydro-α-Galp units), and the concentration of sulfated galactan. Honeycomb-like structures are gradually replaced by more homogeneous networks during the formation of brittle and strong agar-type hydrogels; they are retained, however, in the case of highly elastic carrageenan-type hydrogels. Therefore, the gelation mechanisms should be different for agars and carrageenans. In addition, sulfated galactan gels of high polysaccharide concentration may be structurally different from those of low polysaccharide concentration. Three possible mechanisms of carrageenan/agar gel formation focussing on the specificity of junction zones have been proposed: (i) the double-helical model based on intertwined double helices [217, 218], (ii) the domain model with cation-mediated aggregates of double helices [212, 219], and (iii) the nested, single-helix model with cation-specific salt bridges between ordered chain segments [220, 221]. To date, the step-by-step mechanism of gel formation as well as corresponding conformational transition, i. e., coil-helix or coil-double helix are still unclear [216]. Metal cations may be involved in gel formation depending on the presence and distribution of sulfate semiester groups. The shielding effect of alkali metal cations prevents electrostatic repulsion of sulfate groups in κ-carrageenan gels and thus stabilizes the double helices. Agarose gel is not influenced so much by these cations because of the lack of sulfate groups [222].

Carrageenan films have randomly distributed sets of pores [223]. Their mechanical properties depend on the sulfate/anhydro substitution of carrageenan. Pure κ-carrageenan films are stronger, less flexible, and exhibit a higher tensile strength than pure $\iota$-carrageenan films. The tensile strength of the mixture of κ/λ and κ/$\iota$-carrageenan films decreases with an increasing contribution of κ-carrageenan and is lower than that of pure λ and $\iota$-carrageenan films. Reciprocal relations have been demonstrated for the elongations and water vapor permeability [224]. The gelation properties of carrageenans strongly depend on their structure, first of all on the presence of sulfate semiester and 3,6-anhydro groups. The high density of charged sulfates supports extensive conformation of the carrageenan chain. The presence of the 3,6-anhydro-link in λ-carrageenan allows the α-d-Galp residues to revert to their ${}^1{\mathrm{C}}_4$ conformation, which is necessary for the initial double helix formation required for gelling. Alkali treatment of carrageenans or their precursors during processing increases the gelling properties of the final polysaccharide and the strength of gels due to conversion of α-d-Galp6S to the 3,6-anhydride. As a result, κ-carrageenans and furcellaran (κ/β-carrageenan) with less sulfates but more 3,6-anhydro groups form a firm gel with ${\mathrm{K}}^{+}$. By contrast, more sulfated but less or no anhydrated $\iota$- and λ-carrageenans are slightly affected by these cations; $\iota$-carrageenan needs ${\mathrm{Ca}}^{2+}$ to form soft elastic gels, and λ-carrageenan cannot form gels at all. Hot solutions of κ and $\iota$-carrageenans form gels after cooling of their hot aqueous solutions to $40$–$60\text{°C}$ in the presence of the appropriate metal cations. These gels are stable at room temperature, can be re-melted by heating to $5$–$20\text{°C}$ above the gelling temperature, and re-gel after repeated cooling. Highly flexible carrageenan molecules are able to form helical structures when their concentration increases or temperature decreases. The mechanism of κ-carrageenan gelation in the presence of ${\mathrm{K}}^{+}$ and other alkali metal cations (${\mathrm{Na}}^{+}$, ${\mathrm{Rb}}^{+}$) is thus based on the cation supported aggregation of helical dimers, which consist of two single [225] or double [210] helices.

In any case, a two-step gelation mechanism has been proposed for both $\iota$ and κ-carrageenans [212, 214]. Thermally reversible carrageenan gels exhibit hysteresis or a difference between setting and melting temperatures. The gelation of κ-carrageenan solutions is independent of liquid-liquid phase separation due to the rapid formation of cross-linking points [226]. Furcellaran (κ/β-carrageenan) and κ-carrageenan gels are relatively rigid and undergo syneresis, but $\iota$-carrageenan yields soft and elastic gels with very little tendency to undergo syneresis. In this case, the 2-O-sulfate groups on the 3,6-anhydro-α-d-Galp units prevent the tightly-packed aggregation responsible for the rigidity of κ-carrageenan gels.

Agar is commonly defined as the mixture of neutral agarose and highly sulfated agaropectin. The ratio between these parts depends on the algal source of agar. Agarose demonstrates a much higher gelling ability than amylopectin. Agarose is well soluble in hot water (up to $80\text{°C}$) because it has chains at stiff random coil conformation [227, 228, 229]. Upon cooling below the gelation temperature ($40\text{°C}$), the coils reorder to form helices that subsequently laterally aggregate into a network of thick bundles to form a gel. It is unclear, however, if the proposed high-ordered structure is made of a single helix [228, 229, 230] or a double helix [218]. Agarose gels ($\approx [\%m/v]4$) are typically rigid and turbid, and undergo hysteresis between the melting and setting temperatures. The length of helices suitable for stable association increases with heating. Slow cooling leads to longer helices than rapid cooling, and longer helices promote better aggregation. As a result, the gels formed by slow cooling are stronger and more turbid than those obtained by rapid cooling [231]. The gelation process is followed by liquid–liquid phase separation because the network formation based on aggregated bundles leads to the dilution of solute agarose in the network interspaces [226, 232]. The agar chains form bundles of aggregates, generating a network at the temperature of sol–gel transition. Longer agar chains preferably aggregate, while shorter agar chains are retained in the solute and form isolated aggregates upon further cooling. Reheating leads to easy dissociation of the loose aggregates, whereas bundles of aggregates are thermostable like the whole gel [233].

4.3 Gelling/Film Forming by Fucoidans and Ulvans

In contrast to other phycocolloids, there is a little evidence about the gelling and film forming properties of fucoidans. There are, however, several reports that confirm that this polysaccharide by itself or in a mixture with other polymers is able to form gels and films [234]. Varying amounts of fucoidan in a liquid medium may provide specific rheological properties of the resulting gel.

Green seaweed polysaccharide ulvans are well soluble in water. However, despite their high negative charge, they demonstrate condensed conformation and low intrinsic viscosity in aqueous saline or acidic solutions, which is probably due to the hydrophobic contribution of ${\mathrm{CH}}_3$ in Rhap [156]. This polysaccharide is probably not fully dissolved but forms free condensed spherical nanostructures ($10$–$18\mathrm{nm}$) dispersed in the solvent. At strong alkali conditions (pH $13$) these structures collapse into a dense homogeneous network, which is the basis for strong films and gels. Gelation and film formation at lower pH levels needs divalent cations and/or boric acid. Ulvan forms thermoreversible gel in the presence of boric acid, ${\mathrm{Ca}}^{2+}$, or other divalent metal cations (but not ${\mathrm{Mg}}^{2+}$), and narrow region of pH around $7.5$ [11]. The mechanism of gel formation is unique, very complex, and not well understood. The role of borate and cations in ulvan gelation is unclear. Both these reagents probably promote the aggregation of ulvan nanostructures mentioned above, which are interconnected by more hydrophilic parts of noncondensed polysaccharide chains. Ulvan gels should be stabilized by weak reversible interactions, so borate ester bridges between ulvan macromolecules are improbable [301, 302]. GlossaryTerm

NMR

spectroscopy has not confirmed formation of ulvan borate esters [303]. On the other hand, ${\mathrm{Ca}}^{2+}$ may link borate ester, sulfate semi-ester, and/or carboxylate groups forming junction zones. The unique properties of ulvan gel are interesting for systems of controlled gelation, for example, in drug delivery systems [303, 304].

4.4 Interaction with Other Hydrocolloids

Phycocolloids can interact with other natural and chemically modified hydrocolloids and between each other, forming mixed films, gels, and other structures. Some composite films and gels containing phycocolloids are overviewed in Table 22.11. Among these examples, the alginate–pectin mixtures are interesting due to their ability to form thermoreversible synergistic gels at pH $<3.8$ without the addition of sucrose [246, 247]. The strength and melting points of these gels increase at lower pH and GlossaryTerm

M/G

ratios of alginates. Gelation depends on the sequential distribution of monomeric units in the alginate chain and needs tetrameric or longer G-blocks. Mixtures containing alginates with low GlossaryTerm

M/G

ratio and high methoxy (GlossaryTerm

HM

) pectins show the strongest synergism, the highest storage modulus, and the fastest kinetics of gel formation [245]. By contrast, gels based on alginates with high GlossaryTerm

M/G

ratio and low methoxy (GlossaryTerm

LM

) or low methoxy amidated (GlossaryTerm

LMA

) pectin demonstrate lower storage modulus and slower kinetics. Pectin, but not alginate, affects the network density and the strand characteristics of mixed gels, whereas the homogeneity in the gel microstructure decreases with the addition of alginate content but is independent of the pectin. Specific interactions between hydrocolloids may significantly change the properties of hydrogels. For example, the addition of starch, alginate, or κ-carrageenan decreases the strength of agar gels, while plant seed galactomannans demonstrate marked synergistic effects on the strength of agar and κ carrageenan gels [278, 290, 292].

Tab. 22.11 Composite algal polysaccharide (phycocolloid) films and gels containing other hydrocolloids

Rheological properties of starch colloid systems can be regulated by the addition of fucoidan [297]. At lower concentrations, fucoidan probably damages the junction zones between starch macromolecules, but these polysaccharides are not mutually excluded. By contrast, at higher concentrations, fucoidan may induce considerable mutual exclusion through a phase separation process. Mixed films and gels can be prepared based on the electrostatic interaction between negatively charged phycocolloids and positively charged chitosan or protein. For example, a combination of sodium alginate and gelatine provides synergism in the rheological properties of the resulting mixed gels [276]. Chitosan-alginate films exhibit low solubility, good tensile properties, permeability to water vapor, and biocompatibility, which is important for biomedical applications [259, 260]. The addition of fucoidan to chitosan films increases their mechanical robustness and elasticity, making composite chitosan-fucoidan films more suitable as wound dressings than pure chitosan films [298]. Ulvan-chitosan ionic complex gels are also prepared and characterized as more stable than alginic acid (alginate)-chitosan gel under the appropriate conditions [300].

5 Biological Activities and Medicinal Applications

Besides hydrocolloid properties, both sulfated and carboxylated algal matrix cell wall polysaccharides are known to have various specific biological activities, which are prerequisites of pharmaceutical and medicinal applications. Partially sulfated algal polysaccharides exhibit immunomodulatory, anticoagulant, antithrombotic, antimutagenic, anti-inflammatory, antitumor, antiprotozoal, antimicrobial, and antiviral activities [305]. Generally, these biological activities are related to the structure, composition, and sulfate substitution of polysaccharide macromolecules. Among the activities mentioned above, the immunomodulating and anticoagulant activities seem to be immediate and, therefore, the most important for understanding the mechanism of action. As potential anticoagulants, algal polysaccharides are able to compete with the commonly used heparin. Because they affect the immune system, they may enhance antitumor, antibacterial, and antiviral resistance. In medicaments they can be used as an active substance if they possess the specific biological activity necessary for curing, or as a drug carrier if they are able to interact with an active substance (commonly small or nonpolymeric molecules), and thus improve its activity or provide its delivery to specific parts of the organism.

5.1 Immunomodulation and Related Activities

Immunomodulation is based on the specific enhancement or suppression of the host response with the aim to reach the desired immune effect [306]. This approach allows enhanced natural mechanisms of the immune response without the use of therapeutics like antibiotics. Immunomodulators are substances that affect the immune system. They can stimulate, suppress, or modulate any specific part of the immune system, including both adaptive and innate arms of the immune response [307]. Immunomodulators can be classified into three categories: (i) immunoadjuvants or specific immune stimulants, (ii) nonspecific immunostimulants, and (iii) immunosuppressants. Immunoadjuvants are true modulators that select between various types and mechanisms of immune responses. Thus, these agents are used to design vaccines or enhance their efficacy. Nonspecific immunostimulants enhance the basic level of immune response and are thus used for prophylactic purposes or for strengthening the impaired immune system. Both specific and nonspecific immunostimulants activate immunity directly or indirectly to be effective against cancer cells and various infection agents (prions, viruses, bacteria, or microscopic Eukaryota). Therefore, antitumor, antiprion, antiviral, antimicrobial, antifungal, or antiprotozoal activities of algal polysaccharides are often mediated by immune stimulation. By contrast, immunosuppressants are able to weaken the immune response that is often applied to reduce inflammation in various pathological states. Sulfated algal polysaccharides (fucoidans, galactans, etc.) may stimulate/inhibit cell immunity by activation/inhibition of macrophages and/or other cells of the immune systems and enhance/suppress the production of specific antibodies. Macrophages are critical in regulating innate immunity as well as adaptive immune responses by producing an array of cytokines such as interleukins (GlossaryTerm

IL

) GlossaryTerm

IL

-1β and GlossaryTerm

IL

-6, tumor necrosis factor (GlossaryTerm

TNF

)-α and interferon-γ (GlossaryTerm

INF-γ

), and various types of chemokines such as GlossaryTerm

RANTES

(regulated on activation, normal T-cell expressed and secreted), monocyte chemotactic protein (GlossaryTerm

MCP

)-1, macrophage inflammatory protein (GlossaryTerm

MIP

)-1α, thymus and activation regulated chemokine (GlossaryTerm

TARC

), etc. [308, 309]. Polysaccharide macromolecules may interact with specific membrane receptors of macrophages including Toll-like receptor (GlossaryTerm

TLR

)-4, cluster of differentiation (GlossaryTerm

CD

)-14, complement receptor (GlossaryTerm

CR

)-3 and scavenger receptors (GlossaryTerm

SR

). As a result, the intracellular signaling pathways are promoted, involving mitogen-activated protein kinases (GlossaryTerm

MAPK

) and transcription factors. Activation of macrophages via GlossaryTerm

MAPK

induces the production of both nitric oxide (GlossaryTerm

NO

) and cytokines. Partially, interleukin-12 (GlossaryTerm

IL

-12) stimulates the development of T-cells, which produce interleukin-2 (GlossaryTerm

IL

-2) that, in turn, activates proliferation of natural killer (GlossaryTerm

NK

) cells, representing a first line of defence organism against both primary tumors and metastases. GlossaryTerm

NK

cells produce other cytokines, including interferon (GlossaryTerm

INF

)-γ, which can further provoke macrophages in their stimulation of T-cells via induction of GlossaryTerm

IL

-12. GlossaryTerm

NK

-mediated killing of target cells by apoptosis is facilitated by activation of caspase cascades [310]. Many algal polysaccharides have been reported for their immunomodulatory activities as they stimulate the activity of macrophages. Polysaccharides from the red algae Porphyra yezoensis [311, 312] and Gracilaria verrucosa [313] are reported to have macrophage-stimulating activity in vitro and in vivo. A fucoidan isolated from the sporophyll of brown alga Undaria pinnatifida (Korean Miyeokgui, Japanese Mekabu) stimulated macrophages to produce cytokines (GlossaryTerm

IL

-6, GlossaryTerm

TNF

-α) and chemokines (GlossaryTerm

RANTES

, GlossaryTerm

MIP

-1α) from macrophages and splenocytes [125]. Carrageenan isolated from red algae promotes mice leukocytes to produce GlossaryTerm

TNF

-α [314]. A sulfated water-soluble polysaccharide isolated from the green alga Capsosiphon fulvescens is reported to stimulate macrophage to release GlossaryTerm

TNF

-α and GlossaryTerm

IL

-6, and induce the expression of inducible nitric oxide synthase (GlossaryTerm

iNOS

) and cyclooxygenase-2 (GlossaryTerm

COX-2

). These enzymes are responsible for the production of GlossaryTerm

NO

and prostaglandin E2 (GlossaryTerm

PGE2

), respectively [315]. A sulfated glucuronorhamnan, an ulvan, of the green algae Ulva rigida, is also reported to stimulate macrophage to secrete PGE${}_2$ and induce an increase in GlossaryTerm

COX-2

and NOS-2 expression [316]. Immunomodulation and other biological activities of marine algal polysaccharides are reviewed in Table 22.12.

Tab. 22.12 Biological activities of algal cell wall polysaccharides

5.2 Antitumor and Antimutagenic Activities

The antitumor effect of fucoidans and other sulfated algal polysaccharides that are nontoxic for tumor cells is commonly associated with the following indirect activities:

• 1. The inhibition of tumor cell proliferation

• 2. The stimulation of the apoptosis of tumor cells

• 3. Blocking tumor cell metastasis

• 4. The inhibition of blood vessel formation

• 5. The enhancement of various immune responses [393].

Partially, antitumor and antimetastatic activity of these polysaccharides is mediated by increased GlossaryTerm

NO

production by stimulated macrophages [364], activation of GlossaryTerm

NK

cells via specific cytokines, and/or induction of caspase-dependent cancer cell apoptosis via inhibition of specific GlossaryTerm

MAPK

 [310, 381, 394]. Fucoidan may also activate caspase-independent apoptosis via activation of GlossaryTerm

ROS

-mediated GlossaryTerm

MAPK

and regulation of the Bcl-2 family protein-mediated mitochondrial pathway [395]. Fucoidan from Cladosiphon okamuranus significantly inhibited in vivo and in vitro growth and induced apoptosis of human T-cell leukaemia virus type 1 (GlossaryTerm

HTLV

-1) infected T-cells. This fucoidan also inhibited in vivo growth of model GlossaryTerm

HTLV

-1-induced tumors based on subcutaneously transplanted infected T-cell lines in immune deficient mice [396]. Both Miyeokgui (Undaria pinnatifida sporophyll) and commercial (Fucus vesiculosus) fucoidans showed antitumor activity against four types of cancer, i. e., PC-3 (prostate cancer), GlossaryTerm

HeLa

(cervical cancer), GlossaryTerm

A549

(alveolar carcinoma), and GlossaryTerm

HepG2

(hepatocellular carcinoma) cells [137]. Fucoidans blocked tumor cell adhesion that could be important for antimetastatic effects [397, 398]. Fucoidan from Fucus evanescens produced an inhibitory effect against metastasis and potentiated antimetastatic, but not antitumor, effects of cyclophosphamide [348]. Fucoidans suppress angiogenesis and thus reduce the supply of tumor tissues [399, 400]. Fucoidans stimulate proliferation of macrophages [401] and mediate tumor destruction through Th1 cell and GlossaryTerm

NK

cell responses [380]. Fucoidans activate lymphocytes and macrophages mediated by the production of GlossaryTerm

NO

, ${\mathrm{H}}_2{\mathrm{O}}_2$, and cytokines (GlossaryTerm

TNF

-α, GlossaryTerm

IL

-6), contributing to their effectiveness against tumors [402].

5.3 Anti-Inflammatory Activity

Inflammation is a complex physiological phenomenon that responds to injury, infection, and stress. Anti-inflammatory activity is the ability to reduce inflammation and is used, partially, for pain remedying. Cytokines, such as interleukins GlossaryTerm

IL

-1β, GlossaryTerm

IL

-6, GlossaryTerm

TNF

-α, and GlossaryTerm

NO

have been known to play important roles in pro-inflammatory responses involving various types of cells [393]. GlossaryTerm

NO

is synthesized by inducible nitric oxide synthase (GlossaryTerm

iNOS

) isophorm, which is expressed in response to activation of nuclear factor κB (GlossaryTerm

NF-κB

) and GlossaryTerm

MAPK

induced by various effectors, such as bacterial lipopolysaccharides (GlossaryTerm

LPS

) or Concanvalin A (GlossaryTerm

ConA

). GlossaryTerm

PGE2

, GlossaryTerm

COX-2

, and monocyte chemoattractant protein-1 (GlossaryTerm

MCP-1

) are also related to inflammatory states. Sulfated polysaccharide from Sargassum hemiphyllum significantly reduces secretion of GlossaryTerm

NO

and pro-inflammatory cytokines (GlossaryTerm

IL

-1b, GlossaryTerm

IL

-6, GlossaryTerm

TNF

-α) by GlossaryTerm

LPS

-activated macrophages; it also inhibits GlossaryTerm

LPS

-triggered messenger ribonucleic acid (GlossaryTerm

mRNA

) expressions of GlossaryTerm

IL

-1b, GlossaryTerm

iNOS

, and GlossaryTerm

COX-2

, which may be attributed to the downregulation of NF-jB in the nucleus [373]. Similarly, fucoidan from Ecklonia cava inhibits GlossaryTerm

NO

and GlossaryTerm

PGE2

production by GlossaryTerm

LPS

-stimulated macrophages via the inhibition of enzymes GlossaryTerm

iNOS

and GlossaryTerm

COX-2

 [375]. Microglial activation plays an important role in the pathogenesis of neurodegenerative diseases, and fucoidans significantly inhibit GlossaryTerm

LPS

-stimulated microglial production and GlossaryTerm

mRNA

/protein expression of the mentioned inflammatory factors involving suppression of specific GlossaryTerm

MAPK

 [350, 361]. Mekabu (Undaria pinnatifida sporophyll) fucoidan enhanced Th1 cell response and inhibited GlossaryTerm

Th2

cell response in normal mice, reducing the immunoglobulin E (GlossaryTerm

IgE

) level in mice serum [379]. Fucoidan also induces GlossaryTerm

IL

-10 production in plasma and liver, inhibits the production of GlossaryTerm

ConA

-induced proinflammatory cytokines, and thereby prevents liver injury [352]. Fucoidan improves murine chronic colitis by inhibition of GlossaryTerm

IL

-6 production in colonic epithelial cells [370]. The gastro-protective (anti-ulcer) effect of fucoidan against aspirin-induced ulceration may take place through the prevention of the elevation of proinflammatory cytokines GlossaryTerm

IL

-6 and GlossaryTerm

IL

-12 [353]. The anti-ulcer property of fucoidan might contribute to protecting inflammatory cytokine-mediated oxidative damage to gastric mucosa [354]. The anti-obesity effect of fucoidan is also related to its anti-inflammatory activity. Miyeokgui (Mekabu) fucoidan suppresses adipogenesis via the inhibition of major markers and inflammation-related cytokines in adipocytes. Hence, it may afford some potential to control or reduce obesity [378]. Fucoidan from Fucus vesiculosus can be useful for the prevention or treatment of obesity due to its stimulatory lipolysis [358].

5.4 Activities Against Infection Agents

Marine algal polysaccharides, especially sulfated ones, have attracted considerable attention in recent years for their antiviral activities against animal viruses [325, 403, 404], such as GlossaryTerm

HSV

types 1 and 2 [113, 133, 170, 405], human immunodeficiency virus type-1 (GlossaryTerm

HIV

-1) [406, 407], the influenza virus [408], and the human cytomegalovirus (GlossaryTerm

HCMV

) [133]. Although the use of these polysaccharides and oligosaccharides as drugs is in its infancy, currently, some are already in various phases of clinical trials [409, 410, 411, 412]. An Undaria pinnatifida extract comprising predominantly sulfated galactofucan ($60$–$70\%$ in mass and with relatively higher molecular weight of over $30$–$1000\mathrm{kDa}$) has been patented for its in vivo antiviral activity including GlossaryTerm

HSV

-1 [413]. The antiviral activity of algal sulfated polysaccharides may have a direct or an indirect mechanism, i. e., based on (i) hindering the adsorption of viral particles onto the host cell surface and/or direct inhibition of viral replication, or (ii) activation of immune pathways. Many viruses are able to bind to cell surface proteoglycans (heparan sulfates), which is important for the entrance of viral particles into the cell. Because they have a strong affinity to viruses, algal sulfated polysaccharides may inhibit the adhesion of viral particles onto the cell surface and thus put obstacles into the viral life circle [170]. If the antiviral effect of polysaccharides is significant only at the early stage of the viral replication cycle, viral adsorption is proved. By contrast, antiviral activity after infection confirms that the polysaccharide directly inhibits the viral replication and transition of virions between uninfected and infected cells (virucidal effect). For example, sulfated galactans from red algae Sphaerococcus coronopifolius and Boergeseniella thuyoides inhibit in vitro replication of both GlossaryTerm

HIV

and GlossaryTerm

HSV

-1. However, in the case of the GlossaryTerm

HSV

-1 cycle, these polysaccharides block the adsorption step, while a direct virucidal effect was observed for GlossaryTerm

HIV

-1 replication [341]. Antimicrobial and antiprotozoal activities of algal sulfated polysaccharides may be caused by direct interaction with microorganisms, blocking of microorganism-binding cell receptors, or by modulation of the immune response. For example, fucoidan (sulfated galactofucan) from Undaria pinnatifida sporophyll might inhibit cryptosporidiosis by direct binding to the Cryptosporidium parvum-derived functional mediators in the intestinal epithelial cells of neonatal mice [385]. This polysaccharide also exhibits anti-malarial activity against cultured Plasmodium falciparum parasites in vitro and on Plasmodium berghei-infected mice in vivo [414].

5.5 Anticoagulant and Antithrombotic Activities

The anticoagulant activity of sulfated polysaccharides, including those from algal sources is based on the interactions with plasma cofactors, which are natural inhibitors of coagulation proteases [415]. These interactions are complex and depend on the monosaccharide composition, the molecular weight, and the structure and distribution of sulfate groups. Sulfation is always important for both anticoagulant and antithrombotic activities, but the charge density is not always critical. Commonly anticoagulant activity of algal hydrocolloids, i. e., sulfated galactans, mannans, rhamnans, fucans, or more complex polysaccharides, is described as activated partial thromboplastin time (GlossaryTerm

aPTT

), thrombin time (GlossaryTerm

TT

), prothrombin time (GlossaryTerm

PT

), and antithrombin to anticoagulation factor Xa activities in comparison with corresponding values of heparin obtained at the same in vitro and/or in vivo experimental conditions [416]. The anticoagulant activity of algal sulfated polysaccharides is mainly attributed to thrombin inhibition mediated by antithrombin and/or heparin cofactor II, while other mechanisms have also been proposed. For example, several different fucoidan preparations from various algal species, including Fucus versiculosus [417, 418], Laminaria brasiliensis [417], Ecklonia kurome [418], Ascophyllum nodosum [419], Pelvetia caniculata [420], and the sporophyll of Undaria pinnatifida [384], have been reported for their anticoagulant activity. Fucoidans have potent anticoagulant activity mediated by antithrombin and/or heparin cofactor II [421]. The differences in anticoagulation mechanisms could be explained by the structural diversity of these polysaccharides and the interaction with several target proteases. The driving force for the formation of the sulfated polysaccharide/protein complex is the nonspecific electrostatic interaction between these macromolecules; then the complex is stabilized by other (polar or nonpolar) short-range interactions [422]. Anticoagulant polysaccharide should undergo appropriate conformational transitions to possess distorted ordered conformation suitable for protein binding. Model invertebrate sulfated α-l-fucans and sulfated α-l-galactans of regular linear structure show in vitro anticoagulant activity; some of them also express antithrombotic activity on in vivo models [421]. More complex algal sulfated polysaccharides having similar structural patterns may exhibit similar activities, which are not determined by charge density, but rather depend on monosaccharide composition and sulfation pattern. Partially, disulfated internal monosaccharide residues probably have the highest potential for anticoagulant activity due to their ability to place sulfate groups in spatial positions adequate for interaction with proteins. Slight differences in the proportions and/or distribution of sulfated residues may be critical for the interaction between proteases, inhibitors, and activators of the coagulation system, resulting in a distinct pattern in anti and pro-coagulant activities and in the antithrombotic action of algal sulfated galactans [337]. Sulfated galactans need much longer chains for the anticoagulant effect than heparin. Macromolecules of $\approx$ $15$–$45\mathrm{kDa}$ are able to bind antithrombin but are too short to link the plasma inhibitor and thrombin. Binding to different sites on antithrombin, sulfated galactans are less effective than heparin at promoting antithrombin conformational activation. A different mechanism predominates over the conformational activation of antithrombin in ensuring the antithrombin-mediated anticoagulant activity of the sulfated galactans. Possibly, sulfated galactan connects antithrombin and thrombin, holding the protease in an inactive form. The conformational activation of antithrombin and the consequent formation of a covalent complex with thrombin appear to be less important for the anticoagulant activity of sulfated galactan than for heparin [338]. The ($1\to 3$)-α-l-galactan, but not α-l-fucan, sulfated at O-2 is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II; these two polysaccharides show similar inhibitions when factor Xa is used instead of thrombin [423]. The proportion and/or distribution of 2,3-disulfated α-Galp units along the galactan chain may be a critical structural motif to promote the interaction of the protease with specific protease and coagulation inhibitors [338, 424]. In contrast to the linear fucans from echinoderms and mammalian glycosaminoglycans, which need thrombin inhibitors (antithrombin or heparin cofactor II) for a reasonable anticoagulant effect, branched fucans (fucoidans) from brown algae are direct inhibitors of thrombin [425]. The occurrence of 2,4-di-O-sulfated units is an amplifying motif for 3-linked α-fucan-enhanced thrombin inhibition by antithrombin. If we replace antithrombin by heparin cofactor II, then the major structural requirement for the activity becomes single 4-O-sulfated fucose units, while the presence of 2-O-sulfated fucose residues always inhibits anticoagulant activity [424]. Therefore, the anticoagulant activities of sulfated galactans and fucans are not determined only by their charge density. The structural requirements for interaction of sulfated galactans and sulfated fucans with coagulation cofactors and their target proteases are stereospecific and not merely a consequence of their charge density and sulfate content [423, 424]. The unusual sulfated d-galactan (hybrid carrageenan) from Botryocladia occidentalis demonstrate marked anticoagulant activity via enhanced inhibition of thrombin and factor Xa by antithrombin and/or heparin cofactor II [62]. Disulfated α-Galp units seem to be necessary for effective anticoagulant action, while high negative charge density itself is not so important. Sulfated galactofucan from Spatoglossum schroederi does not show any in vitro anticoagulant activity but demonstrates a potent antithrombotic activity in vivo and strong stimulation of the synthesis of heparan sulfate by endothelial cells. These effects have not bee observed after desulfation [142].

5.6 Antioxidant Activities

Because they are important for living organisms, enzymatic or nonenzymatic oxidative reactions can be destructive in some cases. Many products, including reactive oxygen species (GlossaryTerm

ROS

) and other free radicals are commonly produced by the oxidation of lipids and other biomolecules. These products promote chain reactions that cause the destruction of cellular structures and hence cell death. Antioxidants, in turn, may terminate chain reactions by free radical scavenging. Living organisms, including marine algae, have developed regulator systems based on natural antioxidants, both enzymatic (peroxidases, superoxide dismutase, and catalase) and nonenzymatic (glutathione, vitamins A, C, and E). If such systems do not work well, oxidative stress may occur and lead to various pathological states. Therefore, antioxidants might prevent many diseases, including degenerative ones, and thus promote human health and longevity. Algal antioxidants including sulfated polysaccharides have been reviewed in the context of algal biology and utilization [426]. The antioxidant activity of algal polysaccharides is defined as their ability to inhibit model oxidation processes; some examples are given in Table 22.12. The antioxidant properties of algal polysaccharides and associated compounds are commonly established as (i) GlossaryTerm

DPPH

(2,2-diphenyl-1-picrylhydrazyl) free-radical scavenging, (ii) oxygen radical absorbance capacity (ORAC), (iii) ferric ion reducing antioxidant power (GlossaryTerm

FRAP

) or ferrous ion chelating ability (GlossaryTerm

FCA

), (iv) GlossaryTerm

ABTS

(2,2${}^{\prime }$-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), and (v) GlossaryTerm

ROS

scavenging assays [427]. Structure and sulfation patterns may influence these antioxidant effects of algal polysaccharides. It has been reported that $\iota$-carrageenan demonstrates higher hydroxyl radical scavenging than λ and κ-carrageenans [328]. Alternatively, algal polysaccharides have been tested in accordance with in vitro and in vivo responses to experimental oxidative stress. For example, it has been reported that ulvan-like sulfated polysaccharide from Ulva lactuca stabilizes mitochondrial and microsomal membranes by prevention of the oxidative stress induced by d-GalN [319].

5.7 Medicinal Materials Based on Algal Polysaccharides

Algal cell wall polysaccharides have great potential for medicinal applications because of their biocompatibility with human tissues, their biodegradability, and their nontoxicity. Among these polysaccharides, commercially available, low-cost alginates are known to be the most suitable for the preparation of biomedical materials based on pure and composite films, gels, and fibers [428, 429]. Due to the high water content, porosity, elasticity, permeability, and the ability to create a moist environment, alginate gels are effective in wound healing, drug delivery, and tissue engineering. Partially, calcium alginate hydrogels demonstrate both haemostatic activity due to ${\mathrm{Ca}}^{2+}$ and promotion of the aggregation of platelets and erythrocytes. The hydration, swelling, and erosion behavior of alginate matrices depend on pH, which is also true for drug release mechanisms [430]. As was mentioned above, alginic acid/alginate is able to form two types of gel at acidic and neutral conditions, which is a good prerequisite for the construction of controlled-release systems [431, 432]. Two types of diffusion systems have been prepared based on alginates [431]: (i) the polymer membrane system, in which the drug formulation (solid, suspension, or solution) is inside semipermeable gel-like polymeric capsules, and (ii) the polymer matrix system, in which the drug is homogeneously dispersed in a rate-controlling polymeric matrix of swelling microspheres or conventional tablets. Sodium alginate matrices can effectively sustain drug release even for highly water-soluble drugs [433, 434]. Alginate-based dressings are widely used to treat various surgery wounds [435, 436, 437]. The medicinal effect of calcium alginate as a wound dressing is based on its cation exchange properties [435]. After application, ${\mathrm{Ca}}^{2+}$ of the film/fibre surface is partially replaced by ${\mathrm{Na}}^{+}$ from blood serum, which leads to swelling, partial dissolution of the polysaccharide, and gelation. The effectiveness of this process depends on the structure and source of the alginate. Some properties of alginate films like transparency, thermal stability, water absorption, and swelling behavior improve with the presence of various additives, for example Aloe vera [438]. Composite materials consisting of alginate and other natural or modified polysaccharides have been shown to be potentially useful in drug delivery systems and other medicinal applications [131, 439]. Much fewer references in the literature are devoted to using fucoidan for wound healing. Because it has various biological activities, this polysaccharide is interesting as a component of wound dressing materials. Fucoidan-chitosan films might be a potential treatment system for dermal burns [298]. Local administration of fucoidan films safely reduced post-surgical adhesion in model animals [440]. Fucoidan improves the physical/chemical properties (roughness, hydrophilicity and surface sulfate content) of silk fibroin films, and brings about anticoagulant activity and enhanced endothelial cell affinity to silk fibroin [441]. Thus, fucoidan/silk fibroin films can be applied as blood-contacting biomaterials.

5.8 How to Improve Algal Polysaccharides

There are many ways to improve initially inactive or weakly active natural algal polysaccharides to fit various medicinal applications. First of all, partial degradation of algal polysaccharides leads to obtaining polymeric molecules of lower molecular mass, smaller oligomeric molecules (oligosaccharides), and small molecules (monosaccharides or their fragments). Smaller fragments of algal polysaccharides are more soluble in water, less viscous, and may interact with various biological targets like cell receptors or viral particles. Marine algal polysaccharides or derived oligosaccharides can be also chemically modified by the addition of polar or nonpolar groups. Partially, sulfation of nonsulfated polysaccharides (alginate) or oversulfation of sulfated algal polysaccharides (galactans, fucoidans) is the way to increase the negative charge density of the macromolecules, which may have a beneficial effect on their immunomodulation, anticoagulant, and other properties.

5.9 Partial Degradation

Marine algal polysaccharides can be degraded by the use of chemical or enzymatic treatment. Examples of microbial enzymes and microorganisms that are able to cleavage algal polysaccharides are shown in Table 22.13. Biological functions of oligosaccharides originating from marine algae were reviewed by [454]; some of these activities are similar to the corresponding activities of native polysaccharides. For example, both native algal polysaccharides and their oligomeric fragments may stimulate defence responses and protect plants against various pathogens [455]. However, in other cases, algal oligosaccharides exhibit novel activities that are nontypical for the initial polysaccharides, even the way of cleavage could be important. Partially, alginate oligosaccharides obtained by enzymatic degradation promote growth and influence the fatty acid composition of the biohydrogen producer of the green algae Chlamydomonas reinhardtii, which may significantly enhance biomass production [456]. By contrast, partial acidic hydrolysis of alginate does not lead to products that are able to affect the growth of these algal cells. Alginate oligosaccharides also promote the growth of the microalga Nannochloropsis oculata, which is used as a feed for sea animals and zooplankton [457]. Alginate oligosaccharides stimulate epidermal growth factor (GlossaryTerm

EGF

)-dependent growth of human keratinocytes [458] and vascular endothelial growth factor (GlossaryTerm

VEGF

)-mediated growth and migration of human endothelial cells [459]. Alginate oligosaccharides stimulate the in vivo growth of probiotic bacteria of the genus Bifidobacterium [460]. Alginate oligosaccharides inhibit colonization of Salmonella enteritidis in broiler chickens [461]. Alginate oligomers affect the growth and quality of biofilms, as well as the state of bacterial cells inside and also reduce the tolerance of wound biofilms to antibiotics [462]. Alginate oligosaccharides have a high scavenging $•\mathrm{OH}$ activity, while fucoidan oligosaccharides demonstrate good ${\mathrm{Fe}}^{2+}$ chelation [463]. Low-molecular weight fuco-oligosaccharides ($<3749\mathrm{Da}$) have been prepared by enzymatic degradation of fucoidan (sulfated and acetylated galactofucan) from the Korean Undaria pinnatifida sporophyll [135, 136]. These fucoidan oligomers exhibit strong anticoagulant activities at which GlossaryTerm

APTT

and GlossaryTerm

TT

are significantly prolonged. Unlike intact fucoidan, these fuco-oligosaccharides do not affect GlossaryTerm

PT

. Therefore, the molecular weight and/or sulfate content may be important factors for the anticoagulant activities of fucoidans. Fucoidan oligosaccharides can markedly reduce the arterial blood pressure and plasma angiotensin II of the resting heart rate (GlossaryTerm

RHR

), and the respiration of GlossaryTerm

RHR

has the same change trend as blood pressure [464]. Carrageenan and agar oligosaccharides have also been prepared and tested on various bioactivities. Various biological activities of these oligosaccharides have been observed, and often these activities depend on the molecular weight and degrees/patterns of sulfation. An oligosaccharide ($2\mathrm{kDa}$) obtained from κ-carrageenan effectively inhibits influenza A H1N1 virus replication, as well as GlossaryTerm

mRNA

and protein expression inside the cell [465]. Both κ and λ-carrageenan oligomers demonstrate antitumor activity via a complex modulation of the immune response [466, 467] or organ-mediated defence reactions [445]. The κ-carrageenan oligosaccharide fraction exhibits a relatively higher antitumor activity against the three types of cancer cells (GlossaryTerm

KB

, BGC, and Hela) than the initial polysaccharide [466]. The κ-carrageenan oligosaccharides inhibited in vitro growth of five microorganisms, i. e., Escherichia coli, Staphylococcus aureus, Saccharomyces cerevisiae (maximal effect), Penicillium citrinum, and Mucor sp. [465]. The short agaro-oligosaccharides ($2$–$4$ units) suppress GlossaryTerm

TNF

-α production and GlossaryTerm

iNOS

expression [468], while somewhat longer oligosaccharides ($6$–$8$ units) can elicit a physiological response in red algae [469, 470]. Agar oligosaccharides obtained by enzymatic cleavage demonstrate antioxidative activities in GlossaryTerm

ROS

scavenging ($•\mathrm{OH}$, $•{{\mathrm{O}}_2}^{-}$) and inhibit lipid peroxidation [447]. Sulfated and longer fragments show stronger antioxidant activities than nonsulfated and shorter ones.

Tab. 22.13 Examples of microbial enzymes and microorganisms degrading marine algal polysaccharides

5.10 Chemical Modification

Sulfation (or oversulfation) of marine algal polysaccharides is an effective way of obtaining biologically active derivatives that are especially interesting as potential anticoagulants. The patterns of sulfation are often an important factor of expected biological activity. The inactive partially sulfated and 3,6-anhydro-substituted κ, $\iota$, and θ-carrageenans have been oversulfated regioselectively, and the anticoagulant activity of the derivatives obtained has been studied by GlossaryTerm

aPTT

in vitro assay [471]. The results confirmed that sulfation at O-2 of 3,6-anhydro-α-d-Galp and at O-6 of β-d-Galp increases the anticoagulant activity. The oversulfated κ-carrageenan showed a $30$ times higher anticoagulant activity on doubling the GlossaryTerm

PT

time in comparison with the native polysaccharide [472]. This derivate also gave a threefold enhancement of the glutamic plasminogen (GlossaryTerm

Glu-Plg

) activation by tissue plasminogen activator (GlossaryTerm

t-PA

) or by urokinase (GlossaryTerm

u-PA

), while both native κ-carrageenan and crude heparin were less active. Similarly, oversulfated fucoidan showed four times higher anticoagulant activity in doubling GlossaryTerm

PT

time and gave higher stimulations of GlossaryTerm

Glu-Plg

activation by GlossaryTerm

t-PA

and by GlossaryTerm

u-PA

in comparison to the native fucoidan [473]. Oversulfation of fucoidan also enhances its antiangiogenic and antitumor activities [474]. Similarly what was mentioned above, oligomers of marine algal polysaccharides can be sulfated or oversulfated as well. Alginate oligosaccharides obtained by treatment with alginate lyase from Vibrio sp. $510$ [442] have been sulfated by the formamide-chlorosulfonic acid method [475]. One of the derivatives obtained showed significant inhibition of tumor growth without direct cytotoxic effect, which was proposed to be caused by immunomodulation. Oversulfated, acetylated, and phosphorylated derivatives of κ-carrageenan oligosaccharides have been prepared and their in vitro antioxidant activities tested [476, 477]. In certain antioxidant systems some of these derivatives exhibit higher activities than the initial poly and oligosaccharides. The κ-carrageenan oligosaccharide derivatives inhibit tumor growth and stimulate macrophage phagocytosis and spleen lymphocyte proliferation [478]. Among them, the oversulfated derivative shows the highest antitumor activity expressed both in the decrease of tumor weight and in eliciting GlossaryTerm

NK

cell activity. These effects are significantly higher than those of the unmodified oligosaccharides. There are two ways to obtain novel materials based on marine algal polysaccharides: (i) covalent cross-linking via previously added substituents and/or cross-linking agents (dialdehydes, epichlorohydrin, phenolics, etc.), and (ii) interaction with synthetic polymers or their fragments. Porous scaffolds based on alginate derivative bearing phenolics has been prepared using horseradish peroxidase catalyzing cross-linkage formation between phenolic hydroxyls [479]. These materials have been shown to be suitable for cell immobilization. Cross-linking carrageenan beads (microgels) have been prepared for controlled release delivery systems using epichlorohydrin as the cross-linker [480]. The swelling/shrinking behavior of these cross-linked microgels in saline solutions has shown great potential for the application in food or pharmaceutical products. Highly stretchable and tough hydrogels have been prepared from cross-linked algal polysaccharide and synthetic polymers [481]. Both types of macromolecules form networks based on ionic (alginate) and covalent (polyacrylamide) cross-linkages. Because they contain $\approx$ $90\%$ water, composite gels can be stretched beyond $20$ times their initial length, and notched samples exhibit a stretch of $17$ times. These novel material containing marine algal polysaccharides (cross-linked alginate) significantly enlarge the area of hydrogel applications.